1. Field of the Invention
This invention relates to the microbiological arts and more particularly to improved diagnostic media for use in isolating, identifying, and classifying Enterobacteriaceae.
2. Description of the Prior Art
In the course of metabolizing lactose, E. coli must make two proteins, the first of which, galactoside permease (also known as lactose permease), is responsible for transporting lactose through the bacterial cell envelope and thus permits the accumulation of the substrate lactose within the cell. The second essential protein is betagalactosidase, which cleaves the disaccharide lactose into the monosaccharides glucose and galactose, which are further metabolized.
When grown in the absence of lactose, the normal E. coli cell produces only basal (extremely low) levels of galactoside permease and beta-galactosidase. However, in the presence of lactose, the cells increase synthesis of these two proteins by one hundred to a thousand times. The "induction" of these two proteins in the presence of lactose is thus a critical step in the cell's ability to ferment lactose. Those lactose positive Enterobacteriaceae that lack the regulatory genes necessary for this "induction" and therefore always produce high levels of beta-galactosidase and galactoside permease are called constitutive. The lac operon of E. coli codes for the structural and regulatory genes described above that are responsible for the metabolism of lactose.
The fermentation of lactose (glucose-4-beta-galactoside) resulting in the production of acid and gas has long been a characteristic used in identifying the Enterobacteriaceae. In particular, lactose fermentation is a major characteristic employed in the identification of species such as E. Coli by bacterial taxonomists, water bacteriologists, and, especially, clinical microbiologists.
All three groups of bacteriologists have employed standard media to differentiate between lactose fermenters and non-lactose fermenters as this trait became the major biochemical test in dividing the Enterobacteriaceae into its component members. While such media differ depending upon the particular purpose to which they are put, in general, they have as their characteristic feature the presence of a lactose substrate upon which the microorganism could act coupled with other conventional nutrient carriers such as agar.
With particular reference to clinical microbiologists and their task of identifying various microbes in specimens presented for analysis, the major objective is to differentiate quickly yet reliably between pathogens, which may be the cause of a particular problem, and a patient's normal bacterial flora. Identification and differentiation of Enterobacteriaceae, which include both pathogens and non pathogenic bacteria usually found to be normal flora, rely mainly on an array of biochemical tests. The ability or inability of a particular microorganism to ferment lactose in triple sugar iron (TSI) agar or other indicator media containing lactose is the single most valuable biochemical tool used in distinguishing one member of the Enterobacteriaceae from another. In particular, lactose positiveness is a major aid in distinguishing non pathogenic E. coli from Salmonella and Shigella, the latter pathogens which are for the most part lactose negative.
A significant problem encountered by the clinical microbiologist is that approximately ten percent of all E. coli appear to be lactose negative. When a lactose negative Enterobacteriaceae is found, extensive biochemical tests must be run to assure that the microbe is not Salmonella or Shigella. Thus, much additional work must be undertaken, and delay arises before the proper identification can be made and treatment for the patient prescribed. Thus, the value and validity of the lactose test is materially reduced by the prevalence of lactose negative E. coli.
Accordingly, a primary object of this invention is to develop an improved basis for distinguishing between lactose positive and lactose negative Enterobacteriaceae.
Another object is to provide improved diagnostic media, the use of which significantly reduces the incidence of false lactose negative determinations of E. coli.
A still further object is to provide an improved method for distinguishing between those lactose positive Enterobacteriaceae that produce enzymes of the lac operon constitutively and those inducible strains that have the regulatory genes typical of E. coli.